Topological Mapping of the Cysteine Residues of N-Carbamyl-D-amino-acid Amidohydrolase and Their Role in Enzymatic Activity (original) (raw)
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Journal of Molecular Biology, 2001
The N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) is used on an industrial scale for the production of D-amino acids. The crystal structure of D-NCAase was solved by multiple isomorphous replacement with anomalous scattering using xenon and gold derivatives, and re®ned to 1.95 A Ê resolution, to an R-factor of 18.6 %. The crystal structure shows a four-layer a/b fold with two six-stranded b sheets packed on either side by two a helices. One exterior layer faces the solvent, whereas the other one is buried and involved in the tight intersubunit contacts. A long C-terminal fragment extends from a monomer to a site near a dyad axis, and associates with another monomer to form a small and hydrophobic cavity, where a xenon atom can bind. Site-directed mutagenesis of His129, His144 and His215 revealed strict geometric requirements of these conserved residues to maintain a conformation of a putative catalytic cleft. A region located within this cleft involving Cys172, Glu47, and Lys127 is proposed for D-NCAase catalysis and is similar to the Cys-Asp-Lys site of N-carbamoylsarcosine amidohydrolase. The homologous active-site framework of these enzymes with distinct structures suggests convergent evolution of a common catalytic mechanism.
Journal of Chromatography B, 2011
The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl--alanine amidohydrolase from Agrobacterium tumefaciens (Atcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl-and N-formyl-amino acids to produce different ␣, , ␥ and ␦ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of ␣-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity.
Biochemistry, 2009
The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxydans, and Sco4986 from Streptomyces coelicolor are currently annotated as Daminoacylases or N-acetyl-D-glutamate deacetylases. These three enzymes are 22−34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of Nformyl-D-Xaa, N-acetyl-D-Xaa, N-succinyl-D-Xaa, and L-Xaa-D-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl substituted derivatives of D-glutamate. The best substrates for this enzyme are N-formyl-D-glutamate (k cat /K m = 5.8 × 10 6 M −1 s −1 ), N-acetyl-D-glutamate (k cat /K m = 5.2 × 10 6 M −1 s −1 ) and L-methionine-D-glutamate (k cat /K m = 3.4 × 10 5 M −1 s −1 ). Gox1177 and Sco4986 preferentially hydrolyze N-acyl substituted derivatives of hydrophobic D-amino acids. The best substrates for Gox1177 are N-acetyl-D-leucine (k cat /K m = 3.2 × 10 4 M −1 s −1 ), N-acetyl-D-tryptophan (k cat /K m = 4.1 × 10 4 M −1 s −1 ) and L-tyrosine-D-leucine (k cat /K m = 1.5 × 10 4 M −1 s −1 ). A fourth protein, Bb2785 from B. bronchiseptica, did not have D-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-D-phenylalanine and N-acetyl-D-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the D-glutamate moiety of the inhibitor is ion-paired to Arg-295 while the α-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein.
Journal of Molecular Biology, 2006
N-Acylamino acid racemase (NAAAR) and N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) are important biocatalysts for producing enantiopure a-amino acids. NAAAR forms an octameric assembly and displays induced fit movements upon substrate binding, while D-NCAase is a tetramer that does not change conformation in the presence of a ligand. To investigate the effects of introducing potentially stabilizing S-S bridges in these different multimeric enzymes, cysteine residues predicted to form inter or intra-subunit disulfide bonds were introduced by site-directed mutagenesis. Inter-subunit S-S bonds were formed in two NAAAR variants (A68C-D72C and P60C-Y100C) and two D-NCAase variants (A302C and P295C-F304C). Intra-subunit S-S bonds were formed in two additional NAAAR variants (E149C-A182C and V265C). Crystal structures of NAAARs variants show limited deviations from the wild-type overall tertiary structure. An apo A68C-D72C subunit differs from the wild-type enzyme, in which it has an ordered lid loop, resembling ligand-bound NAAAR. The structures of A222C and A302C D-NCAases are nearly identical to the wild-type enzyme. All mutants with inter-subunit bridges had increases in thermostability. Compared with the wild-type enzyme, A68C-D72C NAAAR showed similar k cat /K m ratios, whereas mutant D-NCAases demonstrated increased k cat /K m ratios at high temperatures (A302C: 4.2-fold at 65 8C). Furthermore, molecular dynamic simulations reveal that A302C substantially sustains the fine-tuned catalytic site as temperature increases, achieving enhanced activity.
Structural Biology of Cysteine Biosynthetic Pathway Enzymes
Amebiasis, 2014
The cysteine biosynthetic pathway is of central importance for the growth, survival, and pathogenicity of the anaerobic protozoan parasite Entamoeba histolytica . This pathway is present across all species but is absent in mammals. Cysteine, the product of this pathway, is the only antioxidative thiol responsible for fi ghting oxidative stress in E. histolytica . Serine acetyl transferase (SAT) and O -acetyl serine sulfhydrylase (OASS) are the two enzymes catalyzing the de novo cysteine biosynthetic pathway. In all organisms in which so far this pathway is known to exist, both these enzymes associate to form a regulatory complex, but in E. histolytica this complex is not formed. The cysteine biosynthetic pathway has been optimized in this organism to adapt to and fulfi ll its cysteine requirements.
Applied and Environmental Microbiology, 2008
An N -carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar At ) has been characterized. βcar At is most active at 30°C and pH 8.0 with N -carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn 2+ , Ni 2+ , and Co 2+ . The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N -carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N -carbamoyl-β-alanine. βcar At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar At is able to produce monosubstituted β 2 - and β 3 -amino acids, showing better catalytic efficiency ( k cat / K m ) f...
Archives of Biochemistry and Biophysics, 1997
corresponding 2-oxo-acids, hydrogen peroxide and am-The holoenzyme form of Rhodotorula gracilis D-monia. The native DAAO from the yeast Rhodotorula amino acid oxidase, an 80-kDa homodimer, reacted gracilis is an 80-kDa homodimer, containing one FAD only to a limited extent with general thiol reagents molecule per 40 kDa subunit (1, 2). The apoprotein of (2,2-dithiodipyridine, 5,5-dithiobis(2-nitrobenzoic acid), R. gracilis DAAO, obtained with high yield in a fully and N-[7-(dimethylamino)-4-methylcoumarinyl]maleireconstitutable form, is a 40-kDa monomer under all mide) (60% residual activity), whereas the monomeric conditions (2). The sequence of the 368 amino acids in apoprotein was completely inactivated and denatured the enzyme, in which 6 cysteines are present, has been by these reagents. To investigate the presence of thiol determined, and recently, limited trypsinolysis studies residue(s) in the active site of the enzyme, the apoprodemonstrated that the 38.3-kDa enzyme monomer is tein was reconstituted with the 8-(methylsulfonyl)catalytically competent (3, 4). DAAO from R. gracilis FAD chemical-affinity probe. Competitive inhibition shows some characteristics which distinguish it from between this analogue and FAD for apoprotein bindthe enzyme purified from pig kidney, e.g., a low degree ing was observed. The covalent attachment of the flaof primary structure similarity (27% of sequence idenvin analogue to the apoprotein was complete after É20 tity), a tight coenzyme binding (K d of 2 1 10 08 M for h of incubation and the flavinylated enzyme, con-FAD), and the rate-limiting step in the kinetic mechataining 8-(cysteinyl)-FAD, was monomeric and inacnism corresponding to the reductive half-reaction, in tive. After HPLC isolation of the flavin-labeled tryptic contrast with the mammalian enzyme, where this step peptides, Cys208 was identified as the only cysteine to is represented by the product released from the oxireact with the FAD analogue. These results show that dized enzyme (2, 3, 5). The properties of the yeast ena single cysteine of R. gracilis D-amino acid oxidase zyme are of interest in view of its biotechnological and reacts with the flavin analogue and that this is located industrial applications. near or at the FAD-binding domain. ᭧ 1997 Academic Press
Applied and Environmental Microbiology, 2005
One of the primary sources of enzyme instability is protein oxidative modification triggering activity loss or denaturation. We show here that the side chain of Cys108 is the main site undergoing stress-induced oxidation in Trigonopsis variabilis D-amino acid oxidase, a flavoenzyme employed industrially for the conversion of cephalosporin C. High-resolution anion-exchange chromatography was used to separate the reduced and oxidized protein forms, which constitute, in a molar ratio of about 3:1, the active biocatalyst isolated from the yeast. Comparative analysis of their tryptic peptides by electrospray tandem mass spectrometry allowed unequivocal assignment of the modification as the oxidation of Cys108 into cysteine sulfinic acid. Cys108 is likely located on a surface-exposed protein region within the flavin adenine dinucleotide (FAD) binding domain, but remote from the active center. Its oxidized side chain was remarkably stable in solution, thus enabling the relative biochemical characterization of native and modified enzyme forms. The oxidation of Cys108 causes a global conformational response that affects the protein environment of the FAD cofactor. In comparison with the native enzyme, it results in a fourfold-decreased specific activity, reflecting a catalytic efficiency for reduction of dioxygen lowered by about the same factor, and a markedly decreased propensity to aggregate under conditions of thermal denaturation. These results open up unprecedented routes for stabilization of the oxidase and underscore the possible significance of protein chemical heterogeneity for biocatalyst function and stability.
High-Level Expression and One-Step Purification of Cyclic Amidohydrolase Family Enzymes
Protein Expression and Purification, 2001
suggesting that enzymes acting on cyclic amide bonds The cyclic amidohydrolase family enzymes, includare structurally and functionally related at molecular ing hydantoinase, dihydropyrimidinase, allantoinase level, primarily participating in nucleotide metabolism, and dihydroorotase, are metal-dependent hydrolases has been accumulated (3-5). Direct experiments on diand play a crucial role in the metabolism of purine and hydroorotase and hydantoinase deduced the metal pyrimidine in prokaryotic and eukaryotic cells. With binding sites and signature sequence of the enzyme (6, the increasing demand for the elucidation of enzyme 7). With this signature sequence and rigidly conserved structures and functions, along with industrial appliregions, recent study on the related family enzymes, cations, the research on the family enzymes has recently been proliferating, but the related enzymes including dihydroorotase, allantoinase, dihydropyrihad been purified conventionally by multistep purifimidinase, and hydantoinase, proposed a cyclic amidocation procedures. Here, we reported the expression hydrolase family (6, 8). Microbial hydantoinase has in Escherichia coli cells of maltose-binding proteinbeen presumed to be a microbial counterpart of the fused family enzymes and their one-step purification. eukaryotic dihydropyrimidinase (EC 3.5.2.2), which is The expression levels of the fusion proteins account involved in the catabolic degradation of pyrimidine and for 20-35% of the total protein in E. coli, allowing also catalyzes the hydrolysis of a variety of hydantoins. approximately 2-3 mg of the purified proteins by affin-Dihydroorotase catalyzes the reversible cyclization of ity chromatography to be obtained per 0.3 L of bactecarbamyl aspartate to form dihydroorotate, the third rial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion step in de novo pyrimidine biosynthesis (9, 10). Allantoiproteins with Factor Xa, in terms of oligomeric strucnase, the first enzyme involved in the degradation of ture, optimal pH, specific activity, and kinetic property, allantoin, which is an intermediate in the catabolic were also conserved as those from the native enzymes. pathway of purines, was studied from the various The availability of the family enzymes to fusion stratsources (11-13). Including these typical family enegy shows potential as a convenient procedure to zymes, the superfamily was further extended by a rerecombinant protein purification and accelerates mote homology search based on the substructure of the structure-function study of the related family urease (4). These findings support the hypothesis that enzymes.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2002
Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.